Liquid chromatographic method and system

ABSTRACT

A chromatographic monitor includes an array of flow cells with individual light sensors that are collectively an array of photodiodes. The output from the photodiodes are multiplexed. To prevent losing information, the photodiodes are each connected to a different one of a plurality of inputs to the multiplexer through a corresponding one of a plurality of circuits that stores energy during the time the one inlet is not connected through the multiplexer to the signal processing circuitry that forms a part of an absorbance monitor. Preferably, the energy storing circuit is a non-switching circuit with low bandwidth and a flat-topped response to an impulse. This improves the signal to noise ratio. A one pole low pass filter with a (1-1/e) Dirac pulse fall time and equal to the multiplex entire cycle repeat time can perform this function and a one pole low pass filter provides satisfactory results. Still better results can be obtained from a three pole, one or two percent overshoot filter with combined minimum frequency bandwidth and fast rise time. A rise time equal to ½ the multiplex entire cycle.

RELATED CASES

This application is a divisional of U.S. patent application Ser. No.11/149,900 filed Jun. 10, 2005, which is a divisional of U.S. patentapplication Ser. No. 10/082,710 filed Feb. 25, 2002, now U.S. Pat. No.6,904,784 entitled LIQUID CHROMATOGRAPHIC METHOD AND SYSTEM by Robert W.Allington, Dale A. Davison and Scott L. Blakley and assigned to the sameassignee as this application; which is a continuation-in-part of U.S.patent application Ser. No. 09/883,968 filed Jun. 19, 2001, now U.S.Pat. No. 6,755,074, entitled LIQUID CHROMATOGRAPHIC METHOD AND SYSTEM byDale A. Davison and Scott L. Blakley and assigned to the same assigneeas this application; which is a continuation-in-part of U.S. patentapplication Ser. No. 09/794,772 filed Feb. 27, 2001, now U.S. Pat. No.6,427,526, entitled LIQUID CHROMATOGRAPHIC METHOD AND SYSTEM by Dale A.Davison and Scott L. Blakley and assigned to the same assignee as thisapplication.

BACKGROUND OF THE INVENTION

This invention relates to liquid chromatographic methods andapparatuses.

Inexpensive liquid chromatographic apparatuses have been developed andare in use, particularly for preparatory chromatography where theemphasis is on quickly obtaining relatively large numbers of largesamples at low cost. Such systems generally include at least one solventreservoir, a multiple pump, a controller, multiple chromatographiccolumns, a collector and usually a multiple detector. Commonly,provision is made for a gradient to be developed and such gradientsystems require at least two solvent reservoirs and some mechanism formixing the solvent from each of the two reservoirs together to form agradient for application to the column. Because of the cost ofindividual detectors, one for each column, the detector may bemultiplexed.

The prior art apparatuses have a disadvantage in that they are not asinexpensive as desired, require a longer period of time than desired forthe separation or have reduced sensitivity due to multiplexing noise.

SUMMARY OF THE INVENTION

Accordingly, it is an object of the invention to provide a novelchromatographic system and method.

It is a still further object of the invention to provide a low-costmethod of providing substantial amounts of solvent to a chromatographicsystem.

It is a still further object of the invention to provide an inexpensivegradient chromatographic system.

It is a still further object of the invention to provide a low-costdetection system equipped to handle a large number of simultaneouslyeluting chromatographic columns.

It is a still further object of the invention to provide an economicalsystem for driving multiple inexpensive pumps while avoiding damage fromexcessively high pressures such as may be caused by blockage of liquidor jamming of any one of the multiple pumps.

It is a still further object of the invention to improve the sensitivityof signal collection from individual sources during multiplexing.

It is a still further object of the invention to provide a technique forefficient but low cost mixing of liquids during pumping of the liquids.

In accordance with the above and further objects of the invention, achromatographic system includes a plurality of pumps, all driventogether by a single pump motor for drawing solvent from solventreservoirs, pumping the solvent through a plurality of columns forseparation of sample, pumping the solvent and solute through a pluralityof detector cells for detecting solute and pumping the solute into afraction collector for collection. The solvent is pulled from thereservoir through a plurality of outlets of a manifold so that aplurality of flow streams may be pulled into the corresponding pluralityof pumps from one or more solvent reservoirs. The pumps may each receivethe combined output of a plurality of different solvent reservoirs incontrolled ratios, and in the preferred embodiment, with multiplecharges of each solvent for each pump cycle to form a gradient and thedifferent solvents in the case of such a gradient are mixed in the pathbetween a flow inlet conduit to the pump and the pump outlet with thepump cylinder and inlet tube being dimensioned to provide adequatemixing during refill of the pump. The ratios of solvents are controlledby a solenoid operated valve in the preferred embodiment. Mixing in thepump cylinders is aided by a rapid refill stroke pulling solvent into anoff-center inlet port of the piston pumps, causing turbulence.

With this arrangement, a single motor is able to drive a multiplicity ofpumps which together can supply a large amount of solvent to a number ofcolumns simultaneously. In the preferred embodiment, at least twodifferent reservoirs pull solvents and different gradients are appliedto at least some columns. However, embodiments in which the same solventis applied to each column is possible and a gradient may be applied tosome columns and a single solvent to others. In one embodiment, thegradient is formed without separate mixers and the mixing is done in thepump and the inlet to the pump and/or other equipment associated withthe system. The inlet to the pump is offset and receives liquid from anarrow coil. The narrow coil aids in the mixing of the two solvents, bystretching out the two solvents into thinner streams transverselyadjacent to each other along the length of the coil. Mixing along thiselongated interface reduces interstream to tension which we have foundto be a barrier to efficient mixing. However, this in itself does notprovide efficient bulk mixing. A fast pump refill cycle causesturbulence from an off-center inlet to the pump to mix the two thinstreams that are next to each other.

In the event of over-pressure in the liquid, which may be indicative ofblockage or jamming in the system, the system senses the over-pressureand compensates by: (1) reducing the flow rate until the pressure isreduced; or (2) by stopping the pump or pumps and providing anindication of over-pressure so the problem can be corrected such as byattaching a tube to drain the cylinder of the pump; or (3) by manuallydisconnecting or removing the malfunctioning portion of the system; or(4) by continuing the motion of the motor and automatically by-passingany column causing that over-pressure for that pump, such as for examplewith a fluid pressure release valve.

The photodiodes are each connected to a different one of a plurality ofinputs to a multiplexer through a corresponding one of a plurality ofcircuits (hereinafter sometimes referred to as multiplex circuit inputmeans) that stores energy during the time the one inlet is not connectedthrough the multiplexer to the signal processing circuitry that forms apart of an absorbance monitor. Preferably, the energy storing circuit isa non-switching circuit with low bandwidth and a flat-topped response toan impulse. This improves the signal to noise ratio. A one pole low passfilter with a (1-1/e) Dirac pulse fall time and equal to the multiplexentire cycle repeat time can perform this function and a one pole lowpass filter provides satisfactory results. Still better results can beobtained from a three pole, one or two percent overshoot filter withcombined minimum frequency bandwidth and fast rise time. A rise timeequal to ½ the multiplex entire cycle repeat time is satisfactory. Inany event, this filter is connected between the photocell and themultiplexer input.

An inexpensive detecting arrangement is utilized that comprises a lightsource which focuses light from a central spot on a lamp for stabilityand selects the frequency of light with a diffraction grating,reflecting the selected light through a slot and onto a plurality oflight conductors. The selected light is transmitted through the lightconductors to flow cells. Each flow cell has within it two light guidesthat are aligned and have a space between them for some of the fluidfrom the chromatographic column to flow. One of the light guides in eachof the flow cells receives light from a corresponding one of the lightconductors and transmits it to the other light guide through theeffluent from the column without intervening focusing means to providelight-guide to light-guide communication in the flow cell through thefluid passing in between the two light guides. The light that is notabsorbed in the flow cell is detected by photodiodes located directlyagainst the receiving light guides.

From the above description, it can be understood that, thechromatographic system and chromatographic method of this invention islow cost and yet provides substantial yield in a short time.

SUMMARY OF THE DRAWINGS

The above noted and other features of the invention will be betterunderstood from the following detailed description when considered withreference to the accompanying drawings in which:

FIG. 1 is a block diagram of a liquid chromatographic system inaccordance with an embodiment of the invention;

FIG. 2 is a simplified partly-schematic, partly-side elevational view ofsolvent reservoirs, manifolds and a purge system used in the embodimentof FIG. 1;

FIG. 3 is a block diagram of a pump array useful in the embodiment ofFIG. 1;

FIG. 4 is a simplified partly-schematic, partly-rear elevational view ofsolvent reservoir manifold and purge system connections used in theembodiment of FIG. 1;

FIG. 5 is an elevational sectional view of a pump array and motor fordriving the pistons for the pumps in the pump array useful in theembodiment of FIG. 1;

FIG. 6 is a sectional view through lines 6-6 of FIG. 5;

FIGS. 7-12 are progressive schematic drawings of an on-off valve,delayed coil and pump in six different positions of operation: (a) FIG.7 being a first position at the start of a refill stroke of the pump;(b) FIG. 8 being a second position in the refill stroke of the pump; (c)FIG. 9 being a third position in the refill stroke of the pump; (d) FIG.10 being a forth position in the refill stroke of the pump; (e) FIG. 11being a fifth position in the refill stroke of the pump; and (f) FIG. 12being a sixth position in the refill stroke of the pump.

FIG. 13 is partly block, partly-schematic diagram of an over-pressuresystem used in an embodiment of the invention;

FIG. 14 is a block diagram of a column and detector array in accordancewith the embodiment of FIG. 1;

FIG. 15 is a schematic diagram of an array of light sources, flow cellsand sensors in accordance with an embodiment of the invention;

FIG. 16 is a fractional enlarged view of a portion of FIG. 15 showinglight inlets to flow cells in accordance with an embodiment of theinvention;

FIG. 17 is a block diagram illustrating the detection of fluid inaccordance with an embodiment of the invention.

FIG. 18 is fragmentary simplified enlarged view of a portion of theembodiment of FIG. 16;

FIG. 19 is a schematic drawing showing a portion of the optical systemin accordance with an embodiment of the invention;

FIG. 20 is a block diagram showing the interconnections between portionsof the preparatory chromatograph of an embodiment of the invention;

FIG. 21 is a flow diagram of a portion of a program utilized in anembodiment of the invention;

FIG. 22 is a flow diagram illustrating the performance of an embodimentof the invention;

FIG. 23 is a flow diagram illustrating the operation of the pressureoverload protection feature of the invention; and

FIG. 24 is another embodiment of a portion of the column and detectorarray including the flow cells, light sensors, a multiplexer, and signalprocessing circuitry for supplying signals to the microprocessor.

DETAILED DESCRIPTION

In FIG. 1, there is shown a block diagram of a preparatory liquidchromatographic system 10 having a pumping system 12, a column anddetector array 14, a collector system 16, a controller 18 and a purgesystem 20. The pumping system 12 supplies solvent to the column anddetector array 14 under the control of the controller 18. The purgesystem 20 communicates with a pump array 34 to purge the pumps and thelines between the pumps and the columns between chromatographic runs.The pump array 34 supplies solvent to the column and detector array 14from which effluent flows into the collector system 16 under the controlof the controller 18. The controller 18 receives signals from detectorsin the column and detector array 14 indicating bands of solute andactivates the fraction collector system 16 accordingly in a manner knownin the art. One suitable fraction collection system is the FOXY® 200fraction collector available from Isco, Inc., 4700 Superior Street,Lincoln, Nebr. 68504.

To supply solvent to the pump array 34, the pumping system 12 includes aplurality of solvent reservoirs and manifolds, a first and second ofwhich are indicated at 30 and 32 respectively, a pump array 34 and amotor 36 which is driven under the control of the controller 18 tooperate the array of pumps 34 in a manner to be described hereinafter.The controller 18 also controls the valves in the pump array 34 tocontrol the flow of solvent and the formation of gradients as the motoractuates the pistons of the reciprocating pumps in the pump array 34simultaneously to pump solvent from a plurality of pumps in the arrayand to draw solvent from the solvent reservoirs and manifolds such as 30and 32.

During this pumping process, the pressure may increase above the amountdesired because of blockage or jamming. If the pressure increases abovea predetermined amount in one or more of the pumps in the pump array 34,there is an automatic correction mechanism for reducing or releasingpressure from at least that one or more pumps to avoid damage. In thepreferred embodiment, the pressure is reduced by reducing the flow rate.If this does not reduce the pressure to an acceptable value, a warningis provided so the operator may correct the problem such as by usingtubing to by-pass the column. For this purpose, the pressure is sensedwith a pressure transducer, and when it exceeds a preset value above therated pressure such as at 55 psi, the pressure release or reductionmechanism starts so the motor 36 may continuously move the pistons upand down without damage. Moreover, valves in the pump array 34 controlthe amount of liquid, if any, and the proportions of liquids fromdifferent reservoirs in the case of gradient operation that are drawninto the pump and pumped from it. The manifolds communicate with thereservoirs so that a plurality of each of the solvents such as the firstand second solvents in the solvent reservoir manifold 30 and 32respectively can be drawn into the array of pumps 34 to permitsimultaneous operation of a number of pumps.

While in the preferred embodiment, an array of reciprocating pistonpumps are used, any type of pump is suitable whether reciprocating ornot and whether piston or not. A large number of different pumps andpumping principles are known in the art and to persons of ordinary skillin the art and any such known pump or pumping principle may be adaptableto the invention disclosed herein with routine engineering in most casesprovided that one motor drives a plurality of pumps. While two solventsare disclosed in the embodiment of FIG. 1, only one solvent may be usedor more than two solvents. Because of the operation of a plurality ofpumps simultaneously driven by a single motor, efficiency and costreduction are obtained by this pumping mechanism.

To process the effluent, the collector system 16 includes a fractioncollector 40 to collect solute, a manifold 42 and a waste depository 44to handle waste from the manifold 42. One or more fraction collectorscommunicate with a column and detector array 14 to receive the solutefrom the columns, either with a manifold or not. A manifold may be usedto combine solute from more than one column and deposit them together ina single receptacle or each column may deposit solute in its ownreceptacle or some of the columns each may deposit solute in its owncorresponding receptacle and others may combine solute in the samereceptacles. The manifold 42 communicates with the column and detectorarray 14 to channel effluent from each column and deposit it in thewaste depository 44. The fraction collector 40 may be any suitablefraction collector such as that disclosed in U.S. Pat. No. 3,418,084 orthe above-identified FOXY fraction collector.

The column and detector array 14 includes a plurality of particularlyeconomical flow cells, a different one of the flow cells communicatingwith each of the columns. The flow cells include within them lightguides positioned so that the effluent flows between them and aroundthem, the light guides being sufficiently close to obtain suitablesensitivity at high light absorbance for a preparatory operation as willbe described hereinafter and the total cross-sectional area of the flowpath and the total volume of flow being sufficient to permit bubbles, ifany, to flow around the light guides so as to avoid distorting thedetection of light.

In FIG. 2, there is shown a partly schematic and partly elevational viewof the first solvent reservoir and manifold 30, the second solventreservoir and manifold 32 and the purge system 20 illustrating themanner in which the manifolds are mounted in a housing 160. The firstsolvent reservoir and manifold 30 includes a first manifold 52 havingone inlet and ten outlets 58A-58J, a conduit 56 and a first solventreservoir 50, which solvent reservoir 50 holds a first solvent 54. Theconduit 56 communicates with the solvent 54 in the solvent reservoir 50on one end and communicates with the interior of the manifold 52 at itsother end. Each of the outlets 58A-58J of the manifold 52 communicatewith the interior of a different one of ten cylinders of the pumps (notshown in FIG. 2) through appropriate valves. Similarly, the secondmanifold 53 communicates with the second solvent 55 in the secondsolvent reservoir 51 through a conduit 57. The manifold 53 has aplurality of outlet conduits 59A-59J which communicate with theinteriors of a corresponding number of the pump cylinders throughappropriate valves as described in more detail hereinafter so that thesolvent from the reservoir 50 and the solvent from the reservoir 51 maybe mixed together in a proportion that is set in accordance with thetiming of the valves.

The purge manifold 96 communicates with a gas source 90 through aconduit 91 and a pressure regulator 92 and the three-way valve 94 tomaintain an appropriate pressure for purging the lines. This manifold 96has ten outlets 98A-98J each communicating with a different one of theten conduits connecting a corresponding one of the corresponding pumpsto a corresponding one of ten corresponding columns to transmit gas backthrough the piston pumps to purge the cylinders of the piston pumps andthe conduits connecting the pumps to the columns. Each of the conduitsconnected to the purge connector arrangement lead to a correspondingpump in the pump array 34 (FIG. 1) which in turn communicates with thecorresponding one of the columns in the column and detector array 14(FIG. 1). One such purge connector arrangement 76E is shown in FIG. 2connected by a conduit 99E to the outlet 98E from the manifold 96 topurge the conduits 68E and 88E.

Between chromatographic runs, the pressurized gas source 90, which iscommonly a source of nitrogen gas, communicates through the pressureregulator 92 and the three-way valve 94 with the manifold 96 to providepurging fluid to each of the corresponding outlets 98A-98J for each ofthe pump and column combinations indicated by the T joints, one of whichis shown at 85E.

With this arrangement, respective ones of the purge conduits 99A-99J(only 99E being shown in FIG. 2 connecting manifold outlet 98E to checkvalve 82E) are connected to apply air or nitrogen gas or other purgingsubstance to the respective ones of the T-joints 80A-80J (80E beingshown in FIG. 2) to purge conduits 68A-68E (68E being shown in FIG. 2)and 88A-88E (88E being shown in FIG. 2) and their corresponding pumpsthrough a corresponding one of the purge connectors 76A-76J (76E beingshown in FIG. 2). Each of the purge connections, such as 76E,corresponds with a corresponding one of the manifold purge outlets98A-98J, the corresponding one of the check valves 82A-82J andcorresponding ones of the conduits 88A-88E. The check valves 82A-82J arearranged to prevent effluent from the pumps from flowing back to themanifold 96 and the electrically operated three way valve 94 permitsselecting the time for purging under the control of the controller 18(FIG. 1). The purge system 20 permits purging of the pumps as well asthe lines between the pumps and the column and detector array 14 and inthe column and detector array 14.

While in the preferred embodiment, the manifolds 52, 53 and 96 each haveten outlet conduits which communicate with ten pump cylinders throughappropriate valves as will be described hereinafter, each could havemore or less than ten outlets. Each of the reservoirs is similar to thereservoir 30 and operates in a similar manner to provide the samesolvent from the same reservoir to a plurality of pump cylinders forsimultaneous pumping of the solvent into a plurality of columns.

In FIG. 3, there is shown a schematic block diagram of a pump array 34having a plurality of piston pump systems 60A-60J and an over-pressurecircuit 83, the piston pump systems 60A-60E, being shown forillustration in FIG. 3 although in the preferred embodiment there areten such pumps each arranged to communicate with corresponding ones ofthe ten outlets from the manifold 52 (FIG. 2) and with correspondingones of the outlets from the manifold 53 (FIG. 2) to pump solvent fromthe reservoirs 50 and 51 (FIG. 2) into corresponding ones of the columns(not shown in FIG. 3). In FIG. 3, four of the pump systems 60A-60D areshown in block form and a fifth 60E is shown in greater detail with theunderstanding that each of the ten pump systems are substantiallyidentical so that the explanation of the pump system 60E is an adequateexplanation for all of the pump systems.

Each of the pump systems communicates with a corresponding one of themanifold outlets 58A-58J and 59A-59J to receive two different solventsfor the purpose of forming a gradient. They may also communicate with asource of purge fluid as indicated by the purge conduits 66A-66J. Withthis arrangement, each of the pumps draws solvent into it from thesolvent reservoirs 50 and 51 (FIG. 2). The solvent flows from the pumpsthrough a corresponding one of the outlets 68A-68J.

The pump system 60E includes the inlet conduit 58E from the firstsolvent reservoir 50 and manifold 52 (FIGS. 1 and 2), the inlet conduit59E from the second solvent reservoir 51 and manifold 53 (FIG. 2), athree-way solenoid valve 70E, a two-way solvent valve 72E, a long flowconduit 73E, a reciprocating piston pump 74E, and a check valve 78E.With this arrangement, the two different solvents from conduit 58E and59E are applied to the pump 74E through a common point connecting thethree-way solenoid valve 70E and the two-way solvent valve 72E. In thepreferred embodiment, two cycles of solvent are applied for each strokeof the piston pump. The size of the cylinder, the size of the flowconduit 73E, the speed of the refill and delivery strokes of the pistonare selected to ensure mixing within the pump 74E and flow conduit 73Eso as to pump a formed gradient through the conduit 86E, through thecheck valve 78E and the outlet conduit 68E to the column and detectorarray 14 (FIG. 1). For this purpose the pump cylinders are in the rangeof one inch to eight inches long. In the preferred embodiment, thecylinders are 3.5 inches long.

To provide two injections or charges of solvent during a refill portionof a pump cycle, the two-way electronically-controlled solvent valve 72Eopens once during each piston refill stroke of the pump 74E and closesduring the delivery portion of the pump cycle. In the preferredembodiment, the two-way solvent valve 72E is a solenoid valve. Toprovide a gradient, the three-way electronically-controlledproportioning valve 70E twice during each refill stroke opens first tothe first solvent reservoir 50 (FIG. 2) and then to the second solventreservoir 51 (FIG. 2) to provide both solvents in two stages for bettermixing. The proportion of the time the valve 70E is open to the firstsolvent reservoir 50 (FIG. 2) and then to the second solvent reservoir51 (FIG. 2) determines the composition of the mixture in the gradient.Both of the solenoid operated valves 70E and 72E are under the controlof the controller 18 to which they are electrically connected. Apressure transducer 81E communicates with the pump outlet through thejoint 80E and is electrically connected to the over-pressure circuit 83through electrical connection 274E as better described in connectionwith FIG. 13 hereinafter.

The over-pressure circuit 83 is electrically connected to the solenoidvalve 72 to controller 18 (FIG. 20) and to the transducer 81E to controlthe time the solenoid valve 72E is open and thus to control the flowrate or motor speed. It receives signals for this purpose from thecontroller 18 through conductors 278 (FIG. 13). The transducer 81E inthe preferred embodiment is a miniature 0-300 psi (pounds per squareinch) transducer available from Dresser Instruments, AshcroftHeadquarters, 250 East Main Street, Strafford Conn. 0661-5145; Telephone(203) 783-6659 as an Ashcroft K8 transducer, although there are othersuitable transducers available.

In FIG. 4, there is shown an elevational view of the backside of thechromatographic system 10, simplified for purposes of explanationincluding the pump array 34 with a plurality of pumps 74A-74J (74F, 74Eand 74D being shown in FIG. 4) with pistons 182E and 182F being drivenby the carriage 174 as will be explained more completely hereinafter.For convenience, three inlets to the pumps 74F, 74E and 74D are shown,with 74E being at the opposite side of the carriage 174 from 74F and 74Eand 74D. The pumps 74F, 74E, and 74D are connected at their inlet portsto respective ones of the flow conduits 73F, 73E and 73D respectively toreceive fluid from corresponding ones of the valves 70F, 70E, and 70D.The valves 70F, 70E and 70D are, in turn, connected to the valves 72F,72E and 72D to receive solvent from respective ones of the valves 72F,72E and 72D connected to respective ones of the outlets of the manifold52 and from respective ones of the outlets of the manifold 53 so thatthe valves 72F, 72E and 72D combine the first and second solvents andpermit them to flow to corresponding ones of the valves 70F, 70E and70D. Similarly, the manifold 96 has its outlets connected tocorresponding ones of the check valves 82A-82J (82E being shown in FIG.4) and of corresponding ones of the T-joints 80A-80J (T-joint 80E beingshown in FIG. 4) within the conduits 86E and 68E (FIG. 3) and its inletconnected to a source of air or nitrogen 91 through the pressureregulator 92 and valve 94 to provide a purging flow of air or nitrogenbetween chromatographic runs.

In FIG. 5, there is shown an elevational sectional view taken throughlines 5-5 of FIG. 6 of the pump array 34 including pumps 74A-74J and thesingle motor 36 which is a Pittman Model GM 14901E161 available fromPittman Division of Penn Engineering, having an address at 343 GodshallDrive, Harleysville, Pa. 19438-0003. The pump array includes a ballscrew 172, a piston rod drive plate 174, a ball nut assembly 176, and acylinder retaining plate 178. With this arrangement, the motor 36 drivesthe ball screw 172 to pull the piston rod drive plate 174 upwardly andpushes it downwardly as the ball screw assembly 172 is rotated by themotor 36. The ball nut assembly 176 is rigidly attached to the pistonrod drive plate 174. As the piston moves, the pump cylinders are held inplace by the cylinder retaining plate 178 so that each of the pumps pumpsimultaneously.

In this view, only pump 74E and the pump 74J are shown, and only thepump 74E will be described in detail with the understanding that each ofthe pumps 74A-74J are substantially the same. The pump 74E includes thepiston rod 180E, the piston 182E, the cylinder 184E, a piston plug 186E,an inlet 188E and an outlet 190E. With this arrangement, the piston rod180E drives the piston 182E within the cylinder 184E. As the piston 182Eis moved downwardly, solvent is pulled through the inlet 188E in thepiston plug 186E at the top of the cylinder 184E and when the piston182E is moved upwardly, fluid is forced from the pump outlet 190E withinthe plug 186E.

In the preferred embodiment, the pumps 74A-74J have a cylinderdisplacement programmable for 5 to 18 ml and pump at pumping ratesbetween 5 to 50 ml/min. The valves 70A-70J twice each refill cycleselect: (1) an open position to first solvent 54 (FIG. 2) or a closedposition in which no solvent flows for 100 percent solvent 54; or (2) anopen position for the first solvent followed by an open position for thesecond solvent 55 for a mixture. These values may vary and are selectedso that a gradient can be formed suitable for preparatory chromatographyto obtain the desired substance. With this arrangement, the time thevalves are open determines the respective amounts of the first andsecond solvents that are injected in that time period so that both thefirst solvent 54 and second solvent 55 are injected into the pumpcylinder 184E in selected amounts twice in each intake stroke of thepump in which the piston plug 186E moves downwardly.

In the refill of a pump cycle portion, because of the length of the flowpaths in the cylinders and in the flow conduits 73D-73F, the cylinderlength and the speed of the refill stroke, the solvents are mixed toform substantially continuous steps of stepped gradient (the gradientmay proceed in steps but each step from a pump cycle is substantiallycontinuous) as the solvent is pulled inwardly. For this purpose, therefill stroke of the piston is at least 3 times faster than the deliverystroke to cause turbulent flow in the cylinder during refill. Thetwo-way valves 72D-72F permit fluid to flow into the cylinder 184Eduring a refill stroke and close the cylinder 184E during a deliverystroke so that the cylinder 184E receives a fixed amount of fluid whichit pumps outwardly. The stroke is controlled by the motor 36 and ballscrew 172 under the control of the controller 18 (FIG. 1). This isacceptable with preparatory chromatography because the demands on thecontinuousness of the flow are not as great as in analyticalchromatography.

The motor 36 is mounted to the housing of the chromatographic system bythe mounting bracket 192 and coupled to the ball screw 172 through thecoupling 194 to rotate the screw rod within the ball screw 172 and thuspull the drive plate 174 upwardly and downwardly. The drive plate 174 isguided in its path by two guide rods 196 and 198 (FIG. 4).

In FIG. 6, there is shown a sectional view through lines 6-6 of FIGS. 4and 5 showing the placement of the cylinders for the pumps 74A-74J asheld within the cylinder retaining plate 178. As shown in this view, theball screw 172 passes through the plate so as to pull upwardly thepiston drive plate 174 in a delivery stroke and move downwardly thepiston drive plate 174 in a pump cylinder filling stroke. The guide rods196 and 198 guide the drive plate upwardly and downwardly.

In FIGS. 7-12 there is shown a developed view of the two way valve 72E,the inlet tubing 73E, and the pump 74E showing six different positionsof the pump which result in mixing of solvents A and B in the preferredembodiment to provide a gradient that is suitable for preparatorychromatography. The diameter of the inlet tubing 73E is selected so asto facilitate mixing of solvents A and B which are inserted one afterthe other into the tubing 73E by proportioning valve 70E to providecharges into the pump chamber. The pump chamber is also sufficientlylong to facilitate mixing. In the preferred embodiment, the tubing 73Ehas a length of 35 inches and should have a length of between 10 inchesand 250 inches and a narrow inner diameter, such as for example 0.085inches. The cylinder 160E is relatively long and narrow, being 3.6inches long with a diameter of 0.612 inches in the preferred embodiment.It should have a length in the range of 3 to 8 inches and a ratio oflength to diameter of between 3 and 8.

The cylinder 160E is shown in FIG. 7, the initial position, against thehead 168E in which blocks flow into the inlet 162E into the tubing 73Eand outflow from the outlet 164E. A short time later, the piston 161 Ehas been withdrawn causing fluid to flow through the inlet 162E which ison one side of the cylinder 160E to cause mixing as a circular currentis formed such as in the eddy current as shown in FIG. 8 at 166E. Stilllater, as shown in FIG. 9, further eddy currents occur in the pumpchamber as the piston continues to withdraw and as shown in FIG. 10still further eddy currents near the piston. The eddy currents result inmixing before the pump stroke of the piston. In FIG. 12, the upwardstroke is beginning in position six and the downward stroke has ended soas to move a relatively well mixed fluid out through the outlet.

During the flow of the two solvents through the coil such as 73E thesolvent-solvent interface between the two solvents is weakened at leastpartly because it is stretched over a longer length of the coil. Severalinterfaces are formed over this length. This reduces the amount ofinertial energy needed at the inlet of the pump for mixing by the eddycurrents. Thus even though a coil is not a good mixer, the combinationof the coil with the turbulence forming inlet to the pump providesunexpectedly good mixing.

In FIG. 13, there is shown a partly-block, partly-schematic drawing ofthe over-pressure circuit 83 having three pressure transducer circuits270A-270C and a pressure control circuit 272. Only the pressuretransducer circuit 270C is shown in detail in FIG. 13, with theunderstanding that all three pressure control circuits 270A-270C aresubstantially the same. The pressure control circuit 272 includes aninput-output circuit 288 and ten drivers, three of which are shown at280A-280C, one for each of the pressure transducers 81A-81J (see FIG. 3for 81E).

To control the flow rate and thus compensate for over-pressureconditions, the input-output circuit 288 receives a binary code on lines278 from the controller 18 (FIGS. 1 and 20) and in response appliessignals to the selected ones of the drivers 280A-280J to control theon-off time of the solenoids 72A-72G (FIG. 4) and thus to control theflow rate from each of the pumps 74A-74J (FIG. 4) through respectiveones of the conduits 86A-86J (86E being shown in FIG. 3).

To supply a signal to the controller 18 indicating pressure, thepressure transducer circuits 270A-270J (only 270A-270C being shown inFIG. 13 with 270C being shown as a schematic diagram) includes thefour-to-one multiplexer 282, the amplifier 284 and the 24 bit analog todigital converter 286 that applies signals through conduits 276C to thecontroller 18 to supply a signal representing four of the transducers.The signals to the controller 18 representing the other six transducersare supplied by the pressure transducer circuits 270A and 270B as shownin FIG. 13. The four-to-one multiplexer 282 receives inputs onconductors 274G-274J from respective ones of the transducers 81G-81J(81E shown in FIG. 3) and applies these signals one at a time to theoperational amplifier 284.

The operational amplifier 284 is connected with a parallel connected1500 pf (picofarads) capacitor and a 732K (kilo-ohm) resistor betweenits output and inverting terminal. The multiplexer 282 is connected tothe inverting and non-inverting terminals of the amplifier 284 through10K resistors 290A and 290B respectively. The non-inverting terminal ofthe operational amplifier 284 is connected to ground through the 232Kresistor 290C.

The output of the operational amplifier 284 is electrically connected tothe input of the 24 bit analog to digital converter 286 as well as toground through reverse resistance of the diode 292C and the 0.2 uf(microfarad) capacitor 292B for spike protection. The output of the 24bit analog to digital converter 286 is connected through the conductor276C to the controller 18. With this structure, data is clocked into thecircuit twice per second from the analog to digital converter 286 byclock pulses from the control electronics and is corrected by offset andgain by a conventional EEPROM (not shown in FIG. 13).

In operation, the pressure limits of the system are set in thecontroller 18. If a pump channel exceeds that limit, then: (1) the pumpflow and data acquisition rate goes to half speed of the original rateby controlling the motor speed (FIG. 3) through the over-pressurecircuit 83 from the controller 18; (2) if the pressure returns to itsset rate, the pump continues the stroke at half rate until the pump isempty; (3) the pump then refills all the pump channels that are beingrun; (4) pumping is resumed at the original programmed flow and dataacquisition rate; (5) if an over-pressure is detected before thecylinders are empty, the pump rate is set to one-quarter its set maximumrate; (6) if the pressure does not return to normal, the motor isstopped and an alarm given until the problem is cured and the operatorindicates a start; and (7) on later pump strokes, the process isrepeated from step 1.

This procedure allows the separation to be completed on all channelsselected. If the over-pressure is due to the transient effect, such assample crashing in on the column, later pump strokes have a strongersolvent and this may clear up the blockage. If the problem is caused bya flow rate that is too high for the solvents in the columns used, therun progresses at a lower than the programmed flow rate to accommodatethe solvent in the column.

If the pump is operating at one-quarter speed and an over-pressurecondition occurs, this indicates a fairly severe plug. Under thiscondition, the controller 18 places the pump under a hold condition andthe operator is signaled. The operator then manually intervenes andcorrects the problem before continuing. The problem may be corrected byreplacing columns causing the problem with tubing so the separation onthe remaining channels can continue and by other repair work.

In FIG. 14, there is shown a schematic diagram of a column and detectorarray 14 having a plurality of columns and detectors, five of which areindicated as 100A-100E, a corresponding plurality of outlet conduits68A-68E; a corresponding plurality of solute outlets 110A-110E; acorresponding plurality of waste outlets 108A-108E from the manifold 42(FIG. 1) and a fraction collector 40. In the preferred embodiment, thereare ten columns and detectors. For illustration, the column anddetectors 100A-100D are shown as a general block whereas the column anddetector 100E is shown in greater detail with the understanding that thecollector and detectors 100A-100D are substantially the same. Moreover,while five collectors and detectors are shown to correspond with theexample being used in this application, more or fewer could readily beused and ten are used in the preferred embodiment.

The collector and detector 100E includes the injector system 102E, acolumn 104E, a detection system 106E, the waste outlet 108E and thesolute outlet 110E. With this arrangement, solvent, whether a gradientor not, flows in the conduit 88E through the injector 102E, a column104E, the flow cell 122E, where solute may be detected and from thereinto the collection system 40 for the collection of solute and thedisposal of waste. The column 104E may be any type of chromatographiccolumn regardless of the mode of operation and it is generally picked inaccordance with the separation problem. In the preferred embodiment thecolumn is the REDISEP disposable column sold by Isco, Inc., 4700Superior Street, Lincoln, Nebr. 68504. It is mounted to either receive asample injection manually from a syringe or automatically from theinjector 102E as well as receiving solvent on the outlet 88E. Its outletflows through the detection system 106E.

The detection system 106E includes a light source 142E, a flow cell122E, a detector 124E and a valve 126E for channeling fluid either tothe waste outlet 44 through conduit 108E or to the collector on outlet110E. The light source 142E hereinafter referred to as the optical benchapplies light from a source common to each of the column and detectorassemblies 100A-100E and applies it through each of the correspondingones of the flow cells including the flow cell 122E and from there tothe corresponding detectors including the detector 191E. The signalreceived indicates the effluent to be channeled to the collector andthat to be channeled to waste for the particular column and detectorsystem.

The injector system 102E includes a solid sample load cartridge 101E anda four-way manual selective valve 103E for controlling the selection ofsample and injection into the column 104E. In the embodiment of FIG. 14,an individual injector system (injector system 102E being shown in FIG.14) is provided for each of the columns although the outlet from oneinjector could go to a manifold to supply the same sample to a pluralityof columns and/or the outlet from one injection cartridge could go to aplurality of injection valves if desired. Similarly, a single fractioncollector 40 is shown but a plurality of such collectors could be usedwith the individual valves connected to more than one collector. Theinjector 102E includes the four-way valve 103E for alternately injectingsample from the sample cartridge 101E and selecting the solvent gradientfrom the outlet 88E from the pumping system. Thus a sample may beinjected and then with a turning of the manual valve 103E thechromatographic run may be initiated. While a manual four-way valve 103Eis shown, automatic injector valves are also available and may beutilized.

In FIG. 15, there is shown a diagrammatic view of an optical bench 120common to all of the flow cells 122A-122J and one reference flow cell122R, having a single stable illuminated spot 131, a diffraction gratingsystem 132 and a multiple pickup system 134 for providing stable lightto each of the flow cells 122A-122J and the reference cells 122R. Theilluminated spot 131 is the bright spot of a deuterium lamp 130. Withthis arrangement, a single small stable spot of light is transmittedonto the diffraction grating system 132 which in turn supplies the lightto the multiple pickup system 134 for transmission through multiplepaths for the multiple light sources such as 142A-142J and 142R for useby the corresponding detectors 124A-124J and 124R and flow cells122A-122J and 122R in the system. The single light source 130 includes asuitable lamp 136, an aspherical condensing mirror 138, a sourceaperture plate 150 and an aspherical focusing mirror 154.

The lamp 136, which in the preferred embodiment is a deuterium lamp,transmits light from its central spot 131 to the condensing mirror 138which reflects the light through a small aperture 152 in the apertureplate 150 to provide a narrow spot of light to the focusing mirror 154for reflection onto a diffraction grating in the diffraction gratingsystem 132. A suitable system of this type is described in greaterdetail in U.S. Pat. No. 5,239,359 except that instead of includingaperture stops to restrict the light to a small flow cell opening, thelight is focused onto a slit 157 in an aperture plate 156 for multiplelight guides 142A-142J and 142R to multiple flow cells 122A-122J and122R. The grating 132 reflects a stable line of light from the centralspot of a selected frequency through a slit 157 in an aperture plate 156mounted to the collar or tubular member 175 within the multiple pickup134.

The aspherical condensing mirror 138 is used to focus an image of the1-mm diameter light source in the deuterium lamp 130 on the UV entranceslit at the monochromator light entrance. The aspherical focusing mirror154 produces a focused anastigmatic slit image, at the wavelengthselected by the diffraction grating 132, on the slit-shaped entranceaperture of an 11-channel fiber optic bundle. Each channel consist ofone, single discrete UV-grade quartz optical fiber of 400 μm diameter.The fiber optic bundle allows a single sample, low cost monochromator tobe used for multiple UV absorbance chromatographic detectors. Thisresults in cost savings in a parallel system.

The diffraction grating 132 is a plain grating with 1200 grooves permillimeter, and disperses the light from the lamp 136. The angle betweenthe diffraction grating 132 and the central light beam coming from theaspherical focusing mirror 154 determines the center wavelength of thelight entering the multiple individual optic fibers in the fiber opticsbundle. The software controls an encoded motor, which actuates thegrating in the monochromator. This allows the computer to control thedetection wavelength used by the system. This encoded motor preciselysets the angle between the aspherical focusing mirror 154 anddiffraction grating 132 by moving an arm to which the diffractiongrating 132 is attached. The diffraction grating 132 swings on an arm tokeep the monochromator focused throughout the wavelength range.

The light travels through the respective optic fibers in the fiber opticbundle. Each optic fiber is coupled to a flow cell, which is the lightexit of the monochromator. A total of eleven individual optical fibersare organized in a nested linear array in the light inlet and fiberoptic bundle to maximize the amount of light to each individual opticalfiber and minimize the difference in light level and wavelength betweenthem. Ten of the optical fibers are coupled to flow cells, which passlight through the chromatographic flow stream and then to measuringdetectors. The reference fibers (eleventh fiber) is near the center ofthe linear array to minimize flicker noise from the deuterium lamp 130.

The multiple pickup 134 includes the aperture plate 156, the opticalfibers 142A-142J and 142R positioned along the slit 157 so that thenarrow slot of light is applied to them. The optical fibers transmit thelight to corresponding ones of the flow cells 122A-122J and 122R witheach of the flow cells including a corresponding light guide describedhereinafter that transmits the light to a matching light guide in theflow cell. The matching light guide receives the light after it haspassed through the effluent and transmits it to photodetectors.

In FIG. 16 there is shown a plan view of the aperture plate 156 having acentral elongated opening or slit 157 within a tubular member 175. Thecentral elongated opening 157 has within it aperture stops 176R,176A-176J each receiving a corresponding one of the light guides 142R,142A-142J for a reference light source and light sources 142A-142J. Thisprovides substantially equal intensity light sources to each of the flowcells 122R, 122A-122J to provide a reference 122R and ten measuring flowcells. In this manner, a stable source of light is reflected ontomultiple light guides 142R, 142A-142J for use by the multiple detectorsand flow cells of the system. The multiple light guides are a fiberoptics bundle.

In FIG. 17, there is shown a block diagram of the flow cells 122A-122E,the detectors 124A-124E and the controller 18 interconnected toillustrate some aspects of the invention that are applicable to the flowcells 122R, 122A-122J and detectors 124R, 124A-124J. As best shown inFIG. 17, the flow cell 122E includes a first light guide 143E, a secondlight guide 140E and the flow path 148E for effluent through the flowcell 122E. As shown in this view, the two light guides 143E and 140E arepositioned adjacent to each other and in close proximity with the flowpath 148E extending around it with sufficient volume to permit bubblesto pass around the space between the light guides 143E and 140E ratherthan blocking the path in the light guides. The light guide 143E is incommunication at one end with the light guide 140E with the fluid in theflow cell 122E and at its opposite end with a photodiode detector 124Eto detect light absorbance within the flow path 148E. This signal isapplied with appropriate buffering to the controller 18.

The controller 18 includes inter alia signal processing circuitry 144forming a part of an absorbance monitor, a recorder 146 and amicroprocessor 147. The signal processing circuitry 144 receives lightfrom the detectors 124A-124E indicating the light that is absorbed andapplies it to the microprocessor 147 which converts it to a logarithmiccurrent. The recorder 146 may be utilized to record the bands ofeffluent but because the application of this chromatographic system isprincipally preparatory the recorder 146 will be unnecessary for mostapplications. The microprocessor 147 may be an Intel 80C196KC availablefrom Intel Corporation, 1501 S. Mopac Expressway, Suite 400, Austin,Tex. 78746.

In FIG. 18 there is shown an enlarged, fragmentary perspective view ofthe flow cell 122E. The distance between the end of the light guide 143Eand the end of the light guide 140E in the flow path 148E isapproximately 0.1 mm (millimeters) in the preferred embodiment andshould be in the range of 0.02 mm to 5 mm. It must be close enough topass light between the two ends without excessive refraction orattenuation to prevent detection and far enough to provide a measure ofabsorbance sufficient to indicate the solute.

In FIG. 19, there is shown a block diagram of a flow cell 122E and thereference flow cell 122R (dry cell with no fluid for reference purposes)connected to a calibration system to establish an absorbance signal,adjusted to provide a zero baseline. As best shown if FIG. 19, the flowcell 122E has within it a light guide 143E, which in the preferredembodiment is a quartz rod, on one side and on the other side anotherquartz rod 140E positioned with its end close to the end of the quartzrod 143E to provide a short space between them for the flow of fluid148E in the flow path 148 and a large area around them for the flow ofthe liquid and any bubbles that may be in it. The quartz rod 143E abutsor nearly abuts the end of the light conductor 142E to receive light fortransmission through the fluid 148E and into the light conductor 142E.Similarly, the flow cell 122R has the light conductor 142R abutting aquartz rod 143R which is inside the flow cell 122R and closely adjacentto the end of another quartz rod 140R for receiving light transmitted bythe quartz rod 143R.

The light transmitted by the quartz rods 140E and 140R is converted toan electrical signal by the photodiode 191E and 191R respectively. Thissignal is conducted through the circuits 181E and 181R respectivelytransmitting it for absorbance in the fluid 148R to the circuit 181. Thespace between light conductors and the quartz light guide and betweenthe photodiode and light guide is as short as possible to permitfocusing in the case of different diameters. If the same diameter, theywould touch but are separated slightly to permit the light from thesmall diameter to expand to the larger diameter or vice versa.

To receive and correct the signal from the flow cell such as 122E withrespect to the reference 148R, the circuit 181 includes the signalreceiving circuits 181E and 181R to receive and process the signal fromthe flow cells such as the flow cell 122E with respect to the referencesignal from the reference flow cell 122R. The signal receiving circuit181E includes a photodiode detector 191E, and amplifier 192E andanalog-to-digital converter 194E and a logarithmic conversion circuit196E.

The photodiode detector 191E abuts the quartz rod 140E to convert theabsorbance signal from the fluid 148E to an electrical signal, which isamplified in the amplifier 192E and converted to a digital signal. Thedigital signal is converted to a logarithmic signal of the receivedsignal in the converter 196E by a standard digital conversion in themicroprocessor and transmitted to one side of a reference signalsubtracter. Similarly, the signal receiving circuit 181R includes aphotodiode detector 191R for receiving the reference signal from thereference flow cell 148R and converting it to an electric signal.

The electric signal is amplified by an amplifier 192R connected to thephotodiode detector 191R and transmitted to the analog-to-digitalconverter 194R which in turn transmits a digital signal representingabsorbance to the logarithmic of the received signal in the converter196E by a standard digital conversion in the microprocessor andtransmitted to one side of a reference signal subtracter. The referencesignal subtracter subtracts the reference signal from the reference flowcell 122R from the absorbance signal from the flow cell 122E, resultingin a signal representing the absorbance which is transmitted to areference off-set circuit 184. The reference off-set circuit 184transmits a signal to a signal zero control circuit 186 that bysubtracting a baseline constant in a manner known in the art andtransmits the corrected absorbance signal through the conductor 188. Inthe preferred embodiment, there is a reference cell of the ten measuringflow cells and the necessary calculations are performed in amicroprocessor.

The flow cells 122R and 122A-122J have a very short pathlength for thelight, which allows very concentrated samples to be monitored. Thisshort pathlength is accomplished by inserting 2 millimeter diameter UVquartz rod light guides 143R, 143A-143J and 140R, 140A-140J into each ofthe corresponding ones of the flow streams 148R, 148A-148J with a verysmall gap between each pair of two rods (typically 0.1 mm). This allowsa very short effective pathlength for the light, while also allowingunrestricted flow to the fluid around the quartz rods. The light guides143R and 140R and light source from an optical fiber 142R is coupled toa blank (dry) flow cell 122R, which passes light to a reference detector191R. The reference detector signal is used for background optical noiseand drift subtraction on the remaining detector channels. For purposesof best noise and drift reduction, the optical fiber used for thereference is not one of the four outermost fibers in the nested array.

The measuring and reference photodiode signals are amplified with linearamplifiers 192R, 192A-192J (192E and 192R being shown in FIG. 19). Thissignal is converted to a digital information with analog-to-digitalconverters 194R, 194A-194J (194E and 194R being shown in FIG. 19). Thesedigital signals are converted to logarithms in the converters 196R,196A-196J (196E and 196R being shown in FIG. 19). Now the referencesignal can be subtracted to compensate for lamp energy variations in thereference signal subtracter 182. Next the baseline offset value issubtracted in the off-set circuit 184. This zeroes out almost allabsorbance due to optical imbalance, including that of refractive index(thermal) gradients in the clean solvent flowing through the system. Thebaseline offset value is determined at the beginning of the separation.The signal at the start of the separation does not contain any solutes.The signal is stored and subtracted from the signal for the duration ofthe separation. This results in the correct absorbance signal. Bothanalog and digital methods of accomplishing these signal conditioningtasks are well known in the art.

Current state of the art in optical fiber technology results in fibersthat have a varying susceptibility to transmission degradation(solarization) in the UV spectrum. It is also desirable to leave the UVlamp on to improve lamp thermal stability and hence detection stability.To satisfy these conflicting requirements, the diffraction grating isprogrammed to focus visible light on the fiber optics bundle at alltimes except when an actual separation is occurring. It is also possibleto move the grating to the far UV (below 100 nm) where the energy outputof the lamp is negligible. This reduces the amount of time the fibersare exposed to UV thereby reducing solarization, greatly increasing thelife of the optical fibers while allowing the lamp to remain on betweenseparations.

In FIG. 20, there is shown a block diagram having the fraction collectordiverter valves 214, the flow cell and detector array 124, thecontroller 18, the pressure transducer 218 and the valve array 212 forthe pumping system. This block diagram illustrates the connectionsbetween the controller 18, the pump drive motor 36, the fractioncollector diverter valves 214, the flow cell and detector array 124, andthe inlet purge and mixing valves 212. As shown in FIG. 20, thecontroller 18 includes inter alia functional components: the pumpcontroller 200 and the valve and detector controller 201. The valvearray 212 includes the pump mixing valves 70, the inlet valves 72 andthe purge valve 94.

As shown in FIG. 20, the pump controller 200 is connected to the seriespump drive 36 and a pressure transducer 218 in a feed-back arrangementsuch as that described in U.S. Pat. No. 5,360,320, the disclosure ofwhich is incorporated herein by reference. Specifically, the feed-backcircuit disclosed in connection with FIGS. 8 and 9 in columns 11, 12, 13and 14 of U.S. Pat. No. 5,360,320 for controlling the pump disclosed inFIG. 4 of that patent is utilized here. The pump controller 200 alsointeracts with the valve and detector controller 201 to control the flowcell and detector array 124 and the fraction collector diverter valves214 for the fraction collector 40 (FIG. 14). The valve and detectorcontroller 201 supplies signals to control the mixing valves 70A-70Jshown collectively at 70, the inlet valves 72A-72J shown collectively at72 and the purge valve 94 of the valve array 212. With this arrangement,the detection of bands to be collected controls the fraction collectorvalves to channel the collection into appropriate containers.

In FIGS. 21, 22 and 23, there are shown flow diagrams illustrating theoperation of the controller 18 under software control having a series ofprogramed steps 230 for initiating the pump fill cycle as shown in FIG.21, a series of steps 232 for forming a gradient in the pump as shown inFIG. 22, and a series of steps 292 for protecting against over-pressureconditions. The series of steps 230 for initiating pump refill operationincludes a start step 234, a clear-registers step 236 for percentage Bsolvent and total volume, a step 238 to move forward in gradient timeuntil one milliliter is delivered except for the percentage found inpercentage solvent B register and the percentage B solvent array andadding one milliliter to total volume, the step 240 of deciding if totalvolume is equal to the refill stroke or the end of the gradient, thestep 242 of adding the percentage B solvent array together and dividingthe two together to get the average percentage of B solvent to totalsolvent for the stroke and calculating the pumps position for switchingthree-way valves and the step 244 for turning on the two-way valve toopen the path to the fluid from the three-way valve and putting the pumpinto the refill mode and start refilling. These steps proceed insuccession as listed above.

As shown by the decision step 240, if the total volume is equal to therefill stroke or the end of the gradient, the step 240 goes to step 242to add all percentage B solvent array values together and divide bytotal volume to get the average of B solvent to total solvent for thestroke and calculating the pumps position for switching the three-wayvalves. If the decision is no at decision step 240 then step 238 isrepeated to move the pistons in the pump array forward in gradient timeuntil one milliliter is delivered except for the percentage found in thepercentage of B solvent to total solvent array and adding one milliliterto total volume.

When the pump is in the refill mode at the end of step 244 and refillinghas started as shown at position 246 (FIG. 21), the program proceeds tostep 248 (FIG. 22). Step 248 is a decision step deciding if the pumpsposition is equal to the position for switching to the A solvent. If itis then the program proceeds to step 250 to switch the three-way valveto solvent A and then returns to position 246. If the decision at step248 is no, then the program proceeds to step 252 to decide if the pumpsposition is equal to the position for switching to the B solvent. If thedecision is yes, then the program proceeds to step 254 to switch thethree-way valve to solvent B and from there back to position 246. If thedecision is no, then the step proceeds to decision step 256 to decide ifthe pump is full or the pump equal to the total volume. If the decisionis no, then the program proceeds to step 246. If the decision at step256 is yes, then the program proceeds to step 258 to turn off thetwo-way valve after which the program ends as shown at step 260.

In FIG.23, there is shown a flow diagram of the program 292 for handlingover-pressure conditions comprising: (1) a subroutine for normalnon-over-pressure operation 267; (2) a subroutine 264 for over-pressureconditions that can be cured by reduced flow rate such as may occur whenthe preset flow rate is too high for the solvent and packing of thecolumns; (3) a subroutine 266 for handling more difficult over-pressureconditions; and (4) a subroutine 270 for stopping the pump in the caseof a serious jam that must be physically corrected.

Under conditions in which the pressure is not beyond the presetpressure, the flow rate is controlled by the subroutine 267 thatincludes: (1) the starting position 246 (FIG. 21); (2) the decision step272 for determining if the pressure is greater than the preset value;(3) the decision step 274 for determining if the flow rate is at itsfull value; (4) the step 280 of setting the flow rate if it is not atfull value; (5) the step 275 of determining if the pressure is above itslimit with the flow rate at full value; and (6) the step of opening thetwo-way valve 276 if the pressure is within limits and ending in thestep 278 (FIG. 22). In the decision steps 272 and 275, if the pressureis greater than the preset value, the subroutine goes to the subroutine264 for mild over-pressure conditions. If not, the subroutine proceedsto decision step 274 to determine if the flow rate is at full value. Ifit is not at full value then it proceeds to the step 280 to increase theflow rate and returns to the decision step 274. When the flow rate is atfull value and the pressure is within limits, the subroutine proceeds tothe step 276 of opening the two-way valve to begin gradient flow.

When there is over-pressure, the subroutine 264 includes the step ofreading the flow rate 282 and the decision step 284 of determining ifthe flow rate is set at full value. If it is set at full value, itproceeds to the step 286 to reduce the flow rate to one-half of fullvalue and then proceeds back to the subroutine 262 which determinesagain if the pressure is within limits.

If the flow rate is not set at full value because it has been reduced toone-half, then the program proceeds to the step 266 which reduces theflow rate to one-quarter the full value and then proceeds back tosubroutine 267. If the pressure is still too high, it proceeds throughsubroutine 264 to subroutine 270 to either complete the run or stop therun and issue an alarm. The subroutine 270 includes the decision step288 of determining if the pressure is greater than the preset value atone-quarter the flow rate, the step 290 of stopping the pumps andissuing an alarm so the operator may cure a serious blockage such as ajamming condition, the step 291 of waiting for the user to signal thatthe problem has been corrected and the step 289 of setting a flag todisable the over-pressure channel. In the decision step 288, if thepressure is greater than the preset value, the program proceeds to thestep 290 to stop the pump and issue an alarm but if it is not greaterthen it proceeds to the subroutine 262. The subroutine 262 permits apump cycle to be completed even if it is at a lower rate.

In operation, a plurality of simple syringe pumps are driven by the samemotor to draw solvent simultaneously and pump the solvent simultaneouslythrough a corresponding plurality of columns for separation and througha plurality of detectors for detecting solute and channeling it into afraction collector for automatic collection. The solvent is pulled fromone or more manifolds so that a plurality of flow streams may be pulledinto the corresponding plurality of pumps from one or more solventreservoirs to form a gradient. In the case of gradient elution, a valveopens to pull a first solvent into the cylinder and then switches topull in a second solvent. In the preferred embodiment, when forming agradient, the pump receives two cycles of flow from two reservoirs sothat a valve will cause solvent to flow from a first reservoir into thepump cylinder and then, except at the starting point of the gradient,from a second cylinder to pull a first charge of solvent and repeatswith the identical amount from the first cylinder and the secondcylinder to form a second charge of solvent.

The solvents are pulled through a flow passageway that is less thanone-tenth the volume of a charge. The flow is mostly in the transitionalstage between laminar flow, gravity, density and turbulent flow in thepassageway. The passageway has a diameter less than one-half of thediameter of a pump cylinder. The force and rate is enough to causeturbulent mixing in the cylinder of the pump. In this manner, thegradient is mixed within the pump cylinder so that a first mixture ispumped from several pumps together into corresponding columns. If thereis an interface between liquids, it is degraded. It is pumped when themotor moves all of the pistons of the syringe pumps upwardly. Thisprocess is repeated but the gradient may gradually change so that in aseries of steps, a gradient is supplied. The flow through the passagewayproduces good axial mixing and poor transverse mixing of flow on a smallscale and the turbulent flow caused in the pump cylinder enhancestransverse mixing and axial mixing on a larger scale. Larger scale inthis specification means one charge into the cylinder has approximatelyone-tenth to one-half of the pump volume and small scale meansone-eighth to one-hundredth pump volume —full displacement being takenas pump volume (18 ml in the preferred embodiment). Between these valuesthe quality of the mixing is proportionately enhanced.

In FIG. 24, there is shown a block diagram of another embodiment of aportion of the column and detector array forming a part of achromatographic monitor including the flow cells 122A-122E (only 122Eand 122R being shown for simplicity), light sensors 191A-191E, amultiplexer 145, one pole low pass filters 192A-192E for storing energyfrom the photocells between read-out stroke times by the multiplexer145, and signal processing circuitry for supplying signals to themicroprocessor 147 through conductor 188. A one pole low pass filterwith a Dirac pulse fall time (1-1/e) equal to the multiplexer groundcycle time is satisfactory. This circuitry is similar to the circuitryof FIG. 17 and identical reference numbers are used for correspondingparts. The photodiodes of the detectors 191A-191E are each connected toa different one of a plurality of inputs to the multiplexer 145 througha corresponding one of a plurality of circuits 192A-192E and 192R thatstore energy during the time the corresponding inlet is not connectedthrough the multiplexer to the signal processing circuitry that forms apart of an absorbance monitor. Preferably the energy storing circuit isa non-switching circuit with low bandwidth and a flat-topped response toan impulse. This improves the signal to noise ratio.

A low pass filter can perform this function and a one pole low passfilter such as shown at 192E and 192F by way of example, providessatisfactory results, about a 6 times increase in signal to noise ratio.Still better results (about twice) can be obtained from a three pole,one or two percent overshoot filter with combined minimum frequencybandwidth and fast rise time such as those described by Jess andSchuessler, in “IEEE Transactions on Circuit Theory (June 1965)” and “Onthe Design of Pulse-Forming Networks” IEEE Transactions on CircuitTheory, Vol. CT-12, No. 3, pp. 393-400, (September 1965). Such filtershave an almost maximum-flat peak output response which optimizes energystorage. The purpose of the energy storing circuit is to provide closeto 100 percent equality over the collection of signals from thephotodetectors with uniform weighing of the signals from different onesof the photodetectors in spite of the dead time for readout caused bythe multiplexer 145, and also to provide faster rise time compared to agiven noise bandwidth. An example of a suitable filter for a ½ to onesecond multiplex cycle time and little response speed degradation, isthe “30.10.10.D” filter on Table II, p. 399 (ibid, September 1965), withall table elements multiplied by a scale factor of 17.06 Although thisseries of all-pole filters is specified from optomality at a bandwidthother than noise bandwidth, it can be seen that the optimality value ofthe elements does not change except by a single scale factor, withrespect to how bandwidth is defined. It can also be shown that thisfilter function is closely optimal for flat-topped pulse response aswell as speed/bandwidth response. Because of impedance problems thesingle-pole stage of the three-pole filter should be connected to thephotocell as in FIG. 24. The two-pole output stage is connected betweenthe one-pole input stage and the multiplexer. The one-pole embodiment isthe same as FIG. 24 without the added two-pole addition to its left.FIG. 24 as modified shows the three-pole embodiment.

While simply designed syringe pumps are used in the preferredembodiment, any other kind of pump may be used. Moreover, only one cycleof flow of liquids into a pump may be used or several may be used.Similarly, it is not necessary for two cycles of the same mixture to beinjected into a pump during each filling of the cylinder but more cyclesor one cycle can be used as programmed. While in the preferredembodiment, a single motor drives all of the pistons, more than onearray of pumps can be utilized with a motor driving a first pluralityand a different motor driving a second plurality.

The columns are simple separation columns and one column is dedicated toeach pump. After flowing through the column, the liquid flows intoinexpensively constructed detectors in which light is applied throughlight guides into the flow cell and received by a light guide from theflow cell. Photodetector diodes are mounted directly against the ends ofthe receiving light guides to receive electrical signals just outside ofthe flow cell. The spacing of the light guides is such as to provideadequate detection for preparatory chromatograph and the flow cell islarge enough so that while it detects absorbance of fluid flowingbetween the light guides, other fluid flows around the light guides sothat if bubbles are formed in the flow cell, they will pass around theguides. The light guides are sufficiently close together so as to notreceive large bubbles but to receive a substantial amount of lightpassed between the two light guides and be able to determine the amountof solute from the light that is absorbed.

A single lamp provides light which is applied to a condensing mirrorfrom a central spot on the lamp and applied through an aperture plate toa focusing mirror which focuses on a diffraction grating positioned toselect an appropriate frequency of light which is stable in a lineapplied to a slot. The plurality of light conductors to be applied todetectors are positioned along the narrow slot to receive stable lightof substantially equal intensity for transmission to the detectors. Thedetected light is applied to a typical signal processing circuitryforming a part of an absorbance monitor which controls a fractioncollector to collect the preparatory fractions. With this arrangement,since a large number of separations is being performed simultaneously, asubstantial number of independent and simultaneous chromatographicseparations can be obtained in a short time.

Although a preferred embodiment of the invention has been described withsome particularity, it is to be understood that the invention may bepracticed other than as specifically described. Accordingly, it is to beunderstood that, within the scope of the appended claims, the inventionmay be practiced other than as specifically described.

1. A multiple channel liquid chromatographic system, comprising: atleast two syringe pumps for pumping solvent in said system wherein eachof said at least two syringe pumps includes a piston and a cylinder; amoving frame attached to at least two pistons of said two syringe pumps,wherein movement of each of the pistons with respect to a correspondingcylinder of said syringe pumps is carried out by the moving frame. atleast one time-proportioning electronically controllable liquid gradientswitching valve; a first mixing means; a second mixing means; saidsecond mixing means being a pump cylinder with an offset inlet thatforms eddy currents in the pump cylinder; the first mixing meansresiding in a fluid flow path between the at least onetime-proportioning electronically controllable liquid gradient switchingvalve and the said at least one of said at least two syringe pumps inletand the second mixing means resides in the cylinder of the at least oneof said at least two syringe pumps downstream of the inlet of the atleast one time-proportioning electronically controllable liquid gradientswitching valve; wherein the fluid flow path between the said at leastone time-proportioning electronically controllable liquid gradientswitching valve and the at least one of said at least two syringe pumpsinlet is a flow passageway sized to produce formation of elongatedstreams of first and second solvents with mixing in the said passageway,which in combination with mixing caused by eddy currents in the pumpcylinder makes each step of the gradient sufficiently flat andreproducible for a desired set of chromatographic separation processes.2. A multiple channel liquid chromatographic system in accordance withclaim 1 wherein the flow passageway has a volume less than one-tenththat of a single charge, wherein the flow passageway has a diameter ofless than one-half the diameter of the pump cylinder; said flowproducing good axial mixing and poor transverse mixing on a small scalecharge and an outlet of said flow passageway injecting into the pumpcylinder where the flow becomes turbulent flow thus enhancing transversemixing and axial mixing on a large scale.
 3. A multiple channel liquidchromatographic system in accordance with claim 2 wherein the flowpassageway has a volume of at least one-tenth that of a single charge;said flow producing good axial mixing on a small scale and an outlet ofsaid flow passageway injecting into the pump cylinder where the flowundergoes enhanced transverse mixing.
 4. A multiple channel liquidchromatographic system in accordance with claim 2 wherein the flowpassageway has a volume of at least one-tenth that of a single chargewherein the distance required for further transverse mixing is small;said flow producing good axial mixing and an outlet of said flowpassageway injecting into the larger diameter pump cylinder where theflow becomes turbulent and undergoes transverse mixing and axial mixing.5. A multiple channel liquid chromatographic system in accordance withclaim 3 in which said at least one time-proportioning electronicallycontrollable liquid gradient switching valve is arranged to produceconsecutive pulses of liquid from at least one of said at least twosources of liquid to a refill inlet at a fluid velocity high enough toinduce turbulent mixing in a space between a head of said piston andthat part of the cylinder not occluded by the piston.
 6. A multiplechannel liquid chromatographic system in accordance with claim 5 furtherincluding means for synchronizing the at least one time-proportioningelectronically controllable liquid gradient switching valve with refillmovement of said piston so that one charge of each desired fluid at adesired volume proportion is deposited in each pump and mixed to form atleast one part of a step of a stepped gradient.
 7. A multiple channelliquid chromatographic system in accordance with claim 6 furtherincluding: first means for shutting off fluid flow between the said pumpand said at least one time-proportioning electronically controllableliquid gradient switching valve during delivery; second means forsynchronizing the at least one time-proportioning electronicallycontrollable liquid gradient switching valve with refill movement ofsaid piston so that one charge of each desired fluid at a desired volumeproportion is deposited in each pump and mixed to form at least one partof a step of a stepped gradient; and control means for repeating thesaid first and second means at consecutively different or same fluidproportions to produce an entire stepped gradient.
 8. A multiple channelliquid chromatographic system in accordance with claim 7 wherein atleast two equal charges of each of two fluids are alternately deliveredto an inlet of at least one of said at least two syringe pumps; said twofluids being mixed in the at least one time-proportioning electronicallycontrollable liquid gradient switching valve during a rapid, energeticrefill, and then delivered as a single step of a step gradient to therest of said system in the order of sample injection device,chromatographic column, and fraction collector.